Izumi Yoshizawa

Critical role of NK cells rather than V alpha 14(+)NKT cells in lipopolysaccharide-induced lethal shock in mice.

Although macrophages play a central role in the pathogenesis of septic shock, NK1+ cells have also been implicated. NK1+ cells comprise two major populations, namely NK cells and Vα14+NKT cells. To assess the relative contributions of these NK1+ cells to LPS-induced shock, we compared the susceptibility to LPS-induced shock of β2-microglobulin (β2m)−/− mice that are devoid of Vα14+NKT cells, but not NK cells, with that of wild-type (WT) mice. The results show that β2m−/− mice were more susceptible to LPS-induced shock than WT mice. Serum levels of IFN-γ following LPS challenge were significantly higher in β2m−/− mice, and endogenous IFN-γ neutralization or in vivo depletion of NK1+ cells rescued β2m−/− mice from lethal effects of LPS. Intracellular cytokine staining revealed that NK cells were major IFN-γ producers. The Jα281−/− mice that are exclusively devoid of Vα14+NKT cells were slightly more susceptible to LPS-induced shock than heterozygous littermates. Hence, LPS-induced shock can be induced in the absence of Vα14+NKT cells and IFN-γ from NK cells is involved in this mechanism. In WT mice, hierarchic contribution of different cell populations appears likely.

Neutrophilia in LFA-1-Deficient Mice Confers Resistance to Listeriosis: Possible Contribution of Granulocyte-Colony-Stimulating Factor and IL-17

LFA-1 (CD11a/CD18) plays a crucial role in various inflammatory responses. In this study, we show that LFA-1−/− mice are far more resistant to Listeria monocytogenes infection than LFA-1+/− mice. Consistent with this, we found the following: 1) the numbers of granulocytes infiltrating the liver were markedly higher in LFA-1−/− mice than in LFA-1+/− mice, 2) increased antilisterial resistance in LFA-1−/− mice was abrogated by depletion of granulocytes, and 3) the numbers of granulocytes in peripheral blood, and the serum levels of both G-CSF and IL-17 were higher in LFA-1−/− mice than in LFA-1+/− mice. Neither spontaneous apoptosis nor survival of granulocytes from LFA-1−/− mice were affected by physiological concentrations of G-CSF. Our data suggest regulatory effects of LFA-1 on G-CSF and IL-17 secretion, and as a corollary on neutrophilia. Consequently, we conclude that increased resistance of LFA-1−/− mice to listeriosis is due to neutrophilia facilitating liver infiltration by granulocytes promptly after L. monocytogenes infection, although it is LFA-1 independent.

Highly biased type 1 immune responses in mice deficient in LFA-1 in Listeria monocytogenes infection are caused by elevated IL-12 production by granulocytes.

LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses. Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1. At the early stage of listeriosis, numbers of IFN-γ producers in the liver and spleen of LFA-1−/− mice were markedly increased compared with heterozygous littermates and Vα14+NKT cell-deficient mice, and NK cells were major IFN-γ producers. Numbers of IL-12 producers were also markedly elevated in LFA-1−/− mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN-γ production by NK cells. Granulocyte depletion diminished numbers of IL-12 producers and IFN-γ-secreting NK cells in the liver of LFA-1−/− mice. Granulocytes from the liver of L. monocytogenes-infected LFA-1−/− mice were potent IL-12 producers. Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis. We assume that highly biased type 1 immune responses in LFA-1−/− mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN-γ secretion by NK cells. In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.

Rapid Development of a Gamma Interferon-Secreting Glycolipid/CD1d-Specific Vα14+ NK1.1− T-Cell Subset after Bacterial Infection

ABSTRACT The phenotypic and functional changes of glycolipid presented by CD1d(glycolipid/CD1d) specific Vα14+ T cells in the liver of mice at early stages of bacterial infection were investigated. After Listeria monocytogenes infection or interleukin-12 (IL-12) treatment, α-galactosylceramide/CD1d tetramer-reactive (α-GalCer/CD1d+) T cells coexpressing natural killer (NK) 1.1 marker became undetectable and, concomitantly, cells lacking NK1.1 emerged in both euthymic and thymectomized animals. Depletion of the NK1.1+ subpopulation prevented the emergence of α-GalCer/CD1d+ NK1.1− T cells. Before infection, NK1.1+, rather than NK1.1−, α-GalCer/CD1d+ T cells coexpressing CD4 were responsible for IL-4 production, whereas gamma interferon (IFN-γ) was produced by cells regardless of NK1.1 or CD4 expression. After infection, IL-4-secreting cells became undetectable among α-GalCer/CD1d+ T cells, but considerable numbers of IFN-γ-secreting cells were found among NK1.1−, but not NK1.1+, cells lacking CD4. Thus, NK1.1 surface expression and functional activities of Vα14+ T cells underwent dramatic changes at early stages of listeriosis, and these alterations progressed in a thymus-independent manner. In mutant mice lacking all α-GalCer/CD1d+ T cells listeriosis was ameliorated, suggesting that the subtle contribution of the NK1.1− T-cell subset to antibacterial protection is covered by more profound detrimental effects of the NK1.1+ T-cell subset.

alpha-GalCer ameliorates listeriosis by accelerating infiltration of Gr-1(+) cells into the liver

α‐Galactosylceramide (α‐GalCer) activates invariant (i)NKT cells, which in turn stimulate immunocompetent cells. Although activation of iNKT cells appears critical for regulation of immune responses, it remains elusive whether protection against intracellular bacteria can be induced by α‐GalCer. Here, we show that α‐GalCer treatment ameliorates murine listeriosis, and inhibits inflammation following Listeria monocytogenes infection. Liver infiltration of Gr‐1+ cells and γ/δ T cells was accelerated by α‐GalCer treatment. Gr‐1+ cell and γ/δ T‐cell depletion exacerbated listeriosis in α‐GalCer‐treated mice, and this effect was more pronounced after depletion of Gr‐1+ cells than that of γ/δ T cells. Although GM‐CSF and IL‐17 were secreted by NKT cells after α‐GalCer treatment, liver infiltration of Gr‐1+ cells was not prevented by neutralizing mAb. In parallel to the numerical increase of CD11b+Gr‐1+ cells in the liver following α‐GalCer treatment, CD11b−Gr‐1+ cells were numerically reduced in the bone marrow. In addition, respiratory burst in Gr‐1+ cells was enhanced by α‐GalCer treatment. Our results indicate that α‐GalCer‐induced antibacterial immunity is caused, in part, by accelerated infiltration of Gr‐1+ cells and to a lesser degree of γ/δ T cells into the liver. We also suggest that the infiltration of Gr‐1+ cells is caused by an accelerated supply from the bone marrow.

Increased resistance of LFA-1-deficient mice to lipopolysaccharide-induced shock/liver injury in the presence of TNF-alpha and IL-12 is mediated by IL-10: A novel role for LFA-1 in the regulation of the proinflammatory and anti-inflammatory cytokine balance

Challenge with low doses of LPS together with d-galactosamine causes severe liver injury, resulting in lethal shock (low dose LPS-induced shock). We examined the role of LFA-1 in low dose LPS-induced shock. LFA-1−/− mice were more resistant to low dose LPS-induced shock/liver injury than their heterozygous littermates, although serum levels of TNF-α and IL-12 were higher in these mice. C57BL/6 mice were not rescued from lethal effects of LPS by depletion of NK1+ cells, granulocytes, or macrophages, and susceptibility of NKT cell-deficient mice was comparable to that of controls. High numbers of platelets were detected in the liver of LFA-1+/− mice after low dose LPS challenge, whereas liver accumulation of platelets was only marginal in LFA-1−/− mice. Following low dose LPS challenge, serum levels of IL-10 were higher in LFA-1−/− mice than in LFA-1+/− mice, and susceptibility to low dose LPS-induced shock as well as platelet accumulation in the liver of LFA-1−/− mice were markedly increased by IL-10 neutralization. Serum levels of IL-10 in LFA-1+/− mice were only marginally affected by macrophage depletion. However, in LFA-1−/− mice macrophage depletion markedly reduced serum levels of IL-10, and as a corollary, susceptibility of LFA-1−/− mice to low dose LPS-induced shock was markedly elevated despite the fact that TNF-α levels were also diminished. We conclude that LFA-1 participates in LPS-induced lethal shock/liver injury by regulating IL-10 secretion from macrophages and that IL-10 plays a decisive role in resistance to shock/liver injury. Our data point to a novel role of LFA-1 in control of the proinflammatory/anti-inflammatory cytokine network.

Role of interleukin-12 in determining differential kinetics of invariant natural killer T cells in response to differential burden of Listeria monocytogenes

Invariant (i) natural killer (NK) T cells are unique T lymphocytes expressing NKR-P1B/C (NK1.1), which recognize glycolipids, notably alpha-galactosylceramide (alpha-GalCer) presented by CD1d. The characteristic phenotype of these iNKT cells undergoes dramatic changes following Listeria monocytogenes infection, and interleukin (IL)-12 is involved in these alterations. Here we show that liver iNKT cells in mice are differentially influenced by the load of infection. Liver alpha-GalCer/CD1d tetramer-reactive (alpha-GalCer/CD1d(+)) T cells expressing NK1.1 became undetectable by day 2 following L. monocytogenes infection and concomitantly cells lacking NK1.1 increased regardless of the severity of infection. Whereas alpha-GalCer/CD1d(+)NK1.1(+) T cells remained virtually undetectable on day 4 following low-dose infection, considerable numbers of these cells were detected in high-dose-infected mice. Whereas numbers of IL-12 producers in the liver on day 4 post infection were comparable in low- and high-dose-infected mice without in vitro restimulation with heat-killed Listeria, those were more prominent in low-dose-infected mice than in high-dose-infected mice after restimulation despite the fact that higher numbers of macrophages and granulocytes infiltrated the liver in high-dose-infected mice than in low-dose-infected mice. Our results indicate that NK1.1 surface expression on iNKT cells is differentially modulated by the burden of infection, and suggest that a high bacterial load probably causes loss of IL-12 production.

Dissociated expression of natural killer 1.1 and T-cell receptor by invariant natural killer T cells after interleukin-12 receptor and T-cell receptor signalling

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with α‐galactosylceramide (α‐GalCer) or interleukin (IL)‐12. Although down‐modulation of surface T‐cell receptor (TCR)/NKR‐P1C (NK1.1) expression has been shown convincingly after stimulation with α‐GalCer, it is unclear whether this also holds true for IL‐12 stimulation. To determine whether failure to detect iNKT cells after IL‐12 stimulation is caused by dissociation/internalization of TCR and/or NKR‐P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL‐12 in the disappearance of iNKT cells after stimulation with α‐GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR‐P1C by iNKT cells after stimulation with α‐GalCer or IL‐12, and the influence of IL‐12 neutralization on the down‐modulation of sTCR/sNKR‐P1C expression by iNKT cells after stimulation with α‐GalCer were examined. The s/cTCR+ s/cNKR‐P1C+ iNKT cells became undetectable after in vivo administration of α‐GalCer, which was partially prevented by IL‐12 neutralization. Whereas s/cNKR‐P1C+ iNKT cells became undetectable after in vivo administration of IL‐12, s/cTCR+ iNKT cells were only marginally affected. mRNA expression of TCR/NKR‐P1C remained unaffected by α‐GalCer or IL‐12 treatment, despite the down‐modulation of cTCR and/or cNKR‐P1C protein expression. By contrast, cTCR+ cNKR‐P1C+ sTCR− sNKR‐P1C− iNKT cells and cNKR‐P1C+ sNKR‐P1C− iNKT cells were detectable after in vitro stimulation with α‐GalCer and IL‐12, respectively. Our results indicate that TCR and NKR‐P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL‐12R. They also suggest that IL‐12 participates, in part, in the disappearance of iNKT cells after stimulation with α‐GalCer by down‐modulating not only sNKR‐P1C, but also sTCR.